Topic 11 Introduction to Separation Science

7/29/2019 Topic 11 Introduction to Separation Science

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SKA6014

ADVANCED ANALYTICAL CHEMISTRY

TOPIC 14Introduction to Separation Science

Azlan Kamari, PhD

Department of ChemistryFaculty of Science and Mathematics

Universiti Pendidikan Sultan Idris


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Introduction to Separations Science

What is separations science?

A collection of techniques for separating complex

mixtures of analytes

Most separations are not an analytical technique in their

own right, until combined with an analytical detector

(often a type of spectrometer)

Key analytical branches:

- chromatography- electrophoresis

- extraction


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What is a Separation?

( a + b + c + d + ) (a) + (b) + ( c ) + (d) +

COMPLETE SEPARATION

( a + b + c + d + ) (a) + ( b + c + d + ..)PARTIAL SEPARATION

( a + b + c + d + ) ( a + b ) + ( b+ a) + ..

ENRICHMENTDETECTION

Separations are key aspects of many modern analytical

methods. Real world samples contain many analytes,

most analytical methods do not offer sufficient selectivity to

be able to speciate all the analytes that might be present.

Most separation methods involve separation of the

analytes into distinct chemical species, followed bydetection:


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Basic Types of Separations

Liquid Column ChromatographyLiquid- Liquid (partition) chromatography (LLC)

stationary and mobile phases (immiscible)

Liquid -Solid (adsorption) chromatography (LSC)

Ion exchange chromatography (IEC)

Exclusion chromatography (EC)

Gas-Liquid chromatography(GLC)

Gas-Solid chromatography(GSC)

Separation Methods Based on Phase Equilibria

Gas-Liquid Gas-Solid Liquid-Liquid Liquid-SolidDistillation Adsorption Extraction Precipitation chrom

Sublimation Gas- Liquid Liq-Liq Chrom Zone meltingFoam Fractionation Molecular sieves Exclusion Fractional crystallization

Ion Exchange

Adsorption

Exclusion

Molecular sieves


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Basic Types of Separations

Separation methods based on rate processes

Barrier Separation Field Separations Other

membrane filtration electrophoresis molecular distillation

dialysis ultracentrifugation enzyme degradation

electro-dialysis thermal diffusion destructive distillation

electro-osmosis electrodeposition

reverse osmosis mass spectrometry

gaseous diffusion

Particle Separation methods

FiltrationSedimentation

Elutriation

Centrifugation

Particle electrophoresis

Electrostatic precipitation


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The 100-Year History of Separations

Russian chemist and botanist Michael Tswett coined theterm chromatography

Chromatography was the first major separation science Tswett worked on the separation of plant pigments,

published the first paper about it in 1903, and tested >100

stationary phases

Separated chlorophyll pigments by their color using CaCO3 (chalk),a polar stationary phase, and petroleum ethers/ethanol/CS2

Mikhail Tswett , Physical chemical studies on chlorophyll adsorptions

Berichte der Deutschen botanischen Gesellschaft24, 316-23 (1906)

Tswetts original adsorption chromatography apparatus


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History of Analytical Chromatography

Chromatography was rediscovered by

Kuhn in 1931, when its analyticalsignificance was appreciated

Chromatography very rapidly gainedinterest:Kuhn (Nobel prize in Chemistry

1937) separates caretenoids and vitamins

1938 and 1939:Karrier and Ruzicka,Nobel prizes in Chemistry

1940:established analytical technique 1948:A. Tiselius, Nobel prize for

electrophoresis and adsorption

1952:A. J. P. Martin and R. L. M. Synge,

Nobel prize for partition chromatography,develop plate theory

1950-1960: Golay and Van Deemterestablish theory of GC and LC

1965: Instrumental HPLC developed

Photographs from www.nobelprize.org

A. Tiselius

R. Kuhn A. J. P. Martin

R. L. M. Synge


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Introduction to Chromatography: Terminology

IUPAC Definition: chromatography is a physical method of

separation in which the components to be separated aredistributed between two phases, one of which is stationary

while the other moves in a definite direction

Stationary phase (SP): common name for the column

packing material in any type of chromatography

Mobile phase (MP): liquid media that continuously flows

through the column and carries the analytes

Analyte: the chemical species being investigated (detectedand quantitatively measured) by an analytical method


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Basic Classification of Chromatographic Methods

Column Chromatography

stationary phase is held in a narrow tube through whichmobile phase is forced under pressure

Liquid chromatography

Mobile phase is a liquid solvent

Gas chromatography

Mobile phase is a carrier gas

Supercritical fluid chromatography

Mobile phase is a supercritical fluid

Planar chromatography stationary phase is supported on a flat plate or in the pores

of a paper (e.g. TLC)


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Separation of a Two-component Mixture

This demonstrates the basic concept of continuous elution


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Chromatograms and Electropherograms

Dead time (volume): the mobile phase holdup time, or the time it takes

for an unretained analyte to reach the detector

A chromatogram or electropherogram shows detector

response to analyte presence/concentration

tM

(tR)A

wB

tM = dead time (a.k.a. t0)

tR = retention time

wB = peak width at base


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A Typical LC Chromatogram

This is a typical HPLC UV-detected chromatogram for a fairly

simple mixture of a drug and a degradation product

Note the upward-sloping baseline (we will explain when we

discuss gradient elution)


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Detector Peaks in Separation Sciences

Peak shapes in separation

sciences are generally Gaussianin nature, reflecting the

fundamental nature of the

processes at work (e.g. diffusion)

In practice, real peaks are

generally slightly asymmetric

Fronting peaks

Tailing peaks

Gaussian

Tailing

Fronting


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A way to characterize chromatographic retention is to

measure the time between injection and the maximum ofthe detector response for the analyte. This parameter,

which is usually called the retention time tR, is inversely

proportional to the eluent flow rate.

Retention time is dictated by physics and chemistry: Chemistry (factors that influence distribution)

stationary phase: type and properties

mobile phase: composition and properties

intermolecular forces Temperature

Physics (flow, hydrodynamics)

mobile phase velocity

column dimensions

Retention Time


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Retention Volume

The product of the retention time and the eluent flow rate

(F) is called the retention volume VRand represents the

volume of the eluent passed through the column while

eluting a particular analyte

Component retention volume VR can be divided into two

parts:

Reduced retention volume, which is the volume of the eluent

that passed through the column while the component was

stuck to the surface.

Dead volume, which is the volume of the eluent that passed

through the column while the component was moving with

the liquid phase.

FtVRR


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Mobile Phase Velocity The average linear velocity of analyte migration (in cm/s)

through a column is obtained by dividing the length of the

packed column (L) by the analytes retention time:

Rt

L

The average linear velocity of the mobile phase is just:

Mt

Lu

Flow rate (mL/min) (F) is commonly used as anexperimental parameter, it is related to the cross sectional

area of the column and its porosity:

02 urF

L = length of column

tR= retention time of analyte

tM= retention time of mobile phase (dead time)

u0= linear velocity at column outlet

= fraction of column volume accessible to liquid


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Retention and Differential Migration in

Chromatography

Note: the arrows represent approximate equilibration

Distribution constant

(partition ratio, partition

coefficient):

KBKA

AM

ASA ccK /

B

M

B

SB ccK /


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Relationship Between Retention Time and

Distribution Constant

soluteofmolestotal

phasemobileinsoluteofmoles u

MMSSSSMM

MM

VcVcu

VcVc

Vcu

/1

1

MS VKVu /1

1

M

S

V

KVk

Need to convert distribution constants into something

measurable first express rate as a fraction of mobilephase velocity:

average linear

velocity of

analyte migration average linearvelocity of MP

Define k:

Substitute indefinition ofK

k

u

1

1

Then substitute

in definitions ofu

and kt

L

t

L

MR

1

1


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This leads to the definition k as the retention factor:

The more universal and fundamental retention parameteris the ratio of the retention volume to the dead volume

The parameterkis also known (especially in the earlier

literature) as the capacity factork'

The Retention Factork

M

MR

M

MR

V

VV

t

ttk

M

R

M

R

V

V

t

tk

kt

L

t

L

MR 1

1

rearrange


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Relative Migration Rates: The Selectivity Factor

A

B

A

B

AR

BR

MAR

MBR

K

K

k

k

t

t

tt

tt

,

,

'

'

)(

)(

Selectivity factor (): the ability of a given stationary

phase to separate two components

is by definition > 1 (i.e. the numerator is always larger

than the denominator)

is independent of the column efficiency; it only

depends on the nature of the components, eluent type,eluent composition, and adsorbent surface chemistry. In

general, if the selectivity of two components is equal to

1, then there is no way to separate them by improving

the column efficiency.


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Band Broadening (Column Efficiency)

After injection, a narrow chromatographic band is

broadened during its movement through the column. The higher the column band broadening, the smaller the

number of components that can be separated in a given

time.

The sharpness of the peak is an indication of theefficiency of the column.


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Separation Efficiency and Peak Width

The peak width is an indication of peak sharpness and, in

general, an indication of the column efficiency . However,the peak width is dependent on a number of parameters :

column length

flow rate

particle size

In absence of the specific interactions or sample

overloading, the chromatographic peak can be

represented by a Gaussian curve with the standard

deviation . The ratio of standard deviation to the peak

retention time /tR is called the relative standard

deviation, which is independent of the flow rate.


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Theoretical Plates

A plate: an equilibration step (or zone) between the

analytes, mobile phase, and stationary phase (comes

from distillation theory)

Number of theoretical plates (N): the number of plates

achieved in a separation (increases with longer columns)

Plate height (H): a measure of the separationefficiency of e.g. the column

Smaller H is better

Also known as HETP (height equivalent to a

theoretical plate)

Measures how efficiently the column is packed

Plate equation:

N

LH


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Calculating Theoretical Plates

The convention today is to describe the efficiency of a

chromatographic column in terms of the plate number N,defined by:

2

RtN

In practice, it is more convenient to measure peak width

either at the base line, or at the half height, and not at

0.609 of the peak height, which actually correspond to 2 .

2

216

B

R

W

tN

2

2/1

2

545.5W

tN R


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Band Broadening Processes

t0

later latest

t1 t2

Non-column Broadening Dispersion of analyte in:

Dead volume of an injector

Volume between injector and column

Volume between column and detector

Column Broadening

Van Deemter and related model


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Band Broadening Theory

Column band broadening originates from three main

sources: multiple paths of an analyte through the column

packing (A)

molecular diffusion (B)

effect of mass transfer between phases (C)

In 1956, J.J. Van Deemter introduced the first equation

which combined all three sources and represented them

as the dependence of the theoretical plate height (H) and

the mobile phase linear velocity (u)

R l ti hi B t Pl t H i ht d


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Relationship Between Plate Height and

Separation Variables

Remember:Mt

Lu

tM= retention time of mobile phase (dead time)

The Van Deemter equation is made up of several terms:

Cuu

BAH


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Van Deemter A Term

The A Term: Eddy diffusion

molecules may travel unequal distances in a packedcolumn bed

particles (if present) cause eddies and turbulence

A depends on size of stationary particles (small is best)

and their packing quality (uniform is best)


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Van Deemter A Term

pdH 2

The first cause of band broadening is differing flow

velocities through the packed column

This may be written as:

In this equation, His the plate height arising from the

variation in the zone flow velocity, dp is the average

particle diameter, and is a constant that is close to

unity

Hgets worse (larger) as the particle diameter increases


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Note: The functional form of the term is B/u

Mobile phase

Van Deemter B Term

The B Term: Longitudinal diffusion

The concentration of analyte is less at the edges of the

band than at the center.

The analyte diffuses out from the center to the edges.

Ifu is high or the diffusion constant of the analyte is low,

the B term has less of a detrimental effect


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Van Deemter B Term

u

D

u

BH m2

In this equation, Dmis the analyte diffusion coefficient in the

mobile phase, is a factor that is related to the diffusion

restriction by the column packing (hindrance factor), and u

is the flow velocity.

The higher the eluent velocity, the lower the diffusion effect on the

band broadening

Molecular diffusion in the liquid phase is about five orders of

magnitude lower than that in the gas phase, thus this effect is limited

for LC, but important for GC

The longitudinal diffusion (along the column long axis) leads

to band broadening of the chromatographic zone. Thisprocess may be described by the equation:


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mobile phase

Stationary phase (SP)analyte attracted onto SP

movement onto SPmovement off SP

Van Deemter C Term

Resistance to Mass Transfer:

The analyte takes a certain amount of time to equilibrate betweenthe stationary phase and the mobile phase

If the velocity of the mobile phase is high, and an analyte has a

strong affinity for the stationary phase, then the analyte in the

mobile phase will move ahead of the analyte in the stationary phase

The band of analyte is broadened The higher the velocity of the mobile phase, the worse the

broadening becomes


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where dp is the particle diameter, dfis the thickness of the film,DMandDS

are the diffusion coefficients of the analyte in the mobile/stationaryphases, and u is the flow velocity

Van Deemter C Term

uD

dkfuD

dkfuCuCHM

p

S

f

MS

22 )(')(

The C term is given by two parts (for MP and SP):

The slower the velocity, the more uniformly analyte

molecules may penetrate inside the particle, and the less

the effect of different penetration on the efficiency.

On the other hand, at the faster flow rates the elution

distance between molecules with different penetration

depths will be high.


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The Combined Van Deemter Equation

uD

dkfu

D

dkf

u

DdH

M

p

S

fmp

22 )(')(22

A B C

The most significant

result is that there is an

optimum eluent flow rate

where the separationefficiency will be the

best, and it is similar for

many compounds

R l i


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Resolution

BA

ARBRsWW

ttR

)()(2

k

kNR

s

11

4

The selectivity factor, , describes the separation of band

centers but does not take into account peak widths. Another

measure of how well species have been separated is providedby measurement of the resolution.

The resolution of two species, A and B, is defined as

Baseline resolution is achieved when Rs = 1.5

The resolution is related to the number of column plates (N), the

selectivity factor () and the average retention factor(k) of A

and B:

I i R l ti


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Improving Resolution

For good resolution in separations,

the three terms can be optimizedPoor

Rs ~ 0.8

Increase k

Rs > 1.5

Increase N

Rs > 1.5

Change

Rs > 1.5

k

kNRs11

4

Change temperature (GC)

Change MP composition (LC)

Increasing N(number of plates)

Lengthen column (GC)

Decrease SP particle size (LC)

Increasing (selectivity factor) Changing mobile phase

Changing column temperature

Changing stationary phase

Increasing k(retention factor)

Topic 11 Introduction to Separation Science

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